RESUMEN
The reduction of water-soluble tetrazolium salts (WSTs) is frequently used to determine the metabolic integrity and the viability of cultured cells. Recently, we have reported that the electron cycler menadione can efficiently connect intracellular oxidation reactions in cultured astrocytes with the extracellular reduction of WST1 and that this menadione cycling reaction involves an enzyme. The enzymatic reaction involved in the menadione-dependent WST1 reduction was found strongly enriched in the cytosolic fraction of cultured astrocytes and is able to efficiently use both NADH and NADPH as electron donors. In addition, the reaction was highly sensitive towards dicoumarol with Kic values in the low nanomolar range, suggesting that the NAD(P)H:quinone oxidoreductase 1 (NQO1) catalyzes the menadione-dependent WST1 reduction in astrocytes. Also, in intact astrocytes, dicoumarol inhibited the menadione-dependent WST1 reduction in a concentration-dependent manner with half-maximal inhibition observed at around 50 nM. Moreover, the menadione-dependent WST1 reduction by viable astrocytes was strongly affected by the availability of glucose. In the absence of glucose only residual WST1 reduction was observed, while a concentration-dependent increase in WST1 reduction was found during a 30 min incubation with maximal WST1 reduction already determined in the presence of 0.5 mM glucose. Mannose could fully replace glucose as substrate for astrocytic WST1 reduction, while other hexoses, lactate and the mitochondrial substrate ß-hydroxybutyrate failed to provide electrons for the cell-dependent WST1 reduction. These results demonstrate that the menadione-mediated WST1 reduction involves cytosolic NQO1 activity and that this process is strongly affected by the availability of glucose as metabolic substrate.
Asunto(s)
Astrocitos/metabolismo , Glucosa/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Sales de Tetrazolio/metabolismo , Vitamina K 3/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Dicumarol/farmacología , Inhibidores Enzimáticos/farmacología , NAD(P)H Deshidrogenasa (Quinona)/antagonistas & inhibidores , Oxidación-Reducción , Ratas Wistar , Sales de Tetrazolio/químicaRESUMEN
The synthesis, characterization, biological activity, and toxicology of sila-ibuprofen, a silicon derivative of the most common nonsteroidal anti-inflammatory drug, is reported. The key improvements compared with ibuprofen are a four times higher solubility in physiological media and a lower melting enthalpy, which are attributed to the carbon-silicon switch. The improved solubility is of interest for postsurgical intravenous administration. A potential for pain relief is rationalized via inhibition experiments of cyclooxygenases I and II (COX-I and COX-II) as well as via a set of newly developed methods that combine molecular dynamics, quantum chemistry, and quantum crystallography. The binding affinity of sila-ibuprofen to COX-I and COX-II is quantified in terms of London dispersion and electrostatic interactions in the active receptor site. This study not only shows the potential of sila-ibuprofen for medicinal application but also improves our understanding of the mechanism of action of the inhibition process.
Asunto(s)
Antiinflamatorios no Esteroideos/metabolismo , Ibuprofeno/química , Silicio/química , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Sitios de Unión , Carbono/química , Dominio Catalítico , Ciclooxigenasa 1/química , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/química , Ciclooxigenasa 2/metabolismo , Humanos , Ibuprofeno/metabolismo , Conformación Molecular , Simulación de Dinámica Molecular , Unión Proteica , Teoría Cuántica , Electricidad EstáticaRESUMEN
ß-lapachone (ß-lap) is reduced in tumor cells by the enzyme NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1) to a labile hydroquinone which spontaneously reoxidises to ß-lap, thereby generating reactive oxygen species (ROS) and oxidative stress. To test for the consequences of an acute exposure of brain cells to ß-lap, cultured primary rat astrocytes were incubated with ß-lap for up to 4 h. The presence of ß-lap in concentrations of up to 10 µM had no detectable adverse consequences, while higher concentrations of ß-lap compromised the cell viability and the metabolism of astrocytes in a concentration- and time-dependent manner with half-maximal effects observed for around 15 µM ß-lap after a 4 h incubation. Exposure of astrocytes to ß-lap caused already within 5 min a severe increase in the cellular production of ROS as well as a rapid oxidation of glutathione (GSH) to glutathione disulfide (GSSG). The transient cellular accumulation of GSSG was followed by GSSG export. The ß-lap-induced ROS production and GSSG accumulation were completely prevented in the presence of the NQO1 inhibitor dicoumarol. In addition, application of dicoumarol to ß-lap-exposed astrocytes caused rapid regeneration of the normal high cellular GSH to GSSG ratio. These results demonstrate that application of ß-lap to cultured astrocytes causes acute oxidative stress that depends on the activity of NQO1. The sequential application of ß-lap and dicoumarol to rapidly induce and terminate oxidative stress, respectively, is a suitable experimental paradigm to study consequences of a defined period of acute oxidative stress in NQO1-expressing cells.
Asunto(s)
Astrocitos/efectos de los fármacos , Dicumarol/farmacología , Inhibidores Enzimáticos/farmacología , Naftoquinonas/efectos adversos , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Glutatión/química , Glutatión/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/antagonistas & inhibidores , Oxidación-Reducción , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The past 20 years have resulted in unprecedented progress in understanding brain energy metabolism and its role in health and disease. In this review, which was initiated at the 14th International Society for Neurochemistry Advanced School, we address the basic concepts of brain energy metabolism and approach the question of why the brain has high energy expenditure. Our review illustrates that the vertebrate brain has a high need for energy because of the high number of neurons and the need to maintain a delicate interplay between energy metabolism, neurotransmission, and plasticity. Disturbances to the energetic balance, to mitochondria quality control or to glia-neuron metabolic interaction may lead to brain circuit malfunction or even severe disorders of the CNS. We cover neuronal energy consumption in neural transmission and basic ('housekeeping') cellular processes. Additionally, we describe the most common (glucose) and alternative sources of energy namely glutamate, lactate, ketone bodies, and medium chain fatty acids. We discuss the multifaceted role of non-neuronal cells in the transport of energy substrates from circulation (pericytes and astrocytes) and in the supply (astrocytes and microglia) and usage of different energy fuels. Finally, we address pathological consequences of disrupted energy homeostasis in the CNS.
Asunto(s)
Encéfalo/metabolismo , Metabolismo Energético/fisiología , Neuroquímica/educación , Estudiantes , Animales , Astrocitos/metabolismo , Congresos como Asunto/tendencias , Humanos , Neuroglía/metabolismo , Neuronas/metabolismoRESUMEN
Brain astrocytes are considered to be highly glycolytic, but these cells also produce ATP via mitochondrial oxidative phosphorylation. To investigate how a metabolic depletion of glucose will affect the metabolism of astrocytes, we applied glucose at an initial concentration of 2 mM to cultured primary astrocytes and monitored the cell viability and various metabolic parameters during an incubation for up to 2 weeks. Already within 2 days of incubation the cells had completely consumed the applied glucose and lactate had accumulated in the medium to a concentration of around 3 mM. During the subsequent 10 days of incubation, the cell viability was not compromised while the extracellular lactate concentration declined to values of around 0.2 mM, before the cell viability was compromised. Application of known inhibitors of mitochondrial metabolism strongly accelerated glucose consumption and initial lactate production, while the lactate consumption was completely (antimycin A or 8-hydroxy efavirenz) and partially (efavirenz, metformin or tyrphostin 23) inhibited which caused rapid and delayed cell toxicity, respectively. The switch from glycolytic glucose metabolism to mitochondrial metabolism during the incubation was neither accompanied by alterations in the specific cytosolic lactate dehydrogenase activity or in the WST1 reduction capacity nor in the mitochondrial citrate synthase activity, but a cellular redistribution of mitochondria from a perinuclear to a more spread cytoplasmic localization was observed during the lactate consumption phase. These results demonstrate that cultured astrocytes survive a metabolism-induced glucose depletion very well by consuming lactate as fuel for mitochondrial ATP generation.
Asunto(s)
Astrocitos/metabolismo , Glucosa/metabolismo , Metformina/farmacología , Mitocondrias/efectos de los fármacos , Animales , Astrocitos/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Glucólisis/efectos de los fármacos , Glucólisis/fisiología , Ácido Láctico/metabolismo , Mitocondrias/metabolismo , Ratas WistarRESUMEN
Cellular reduction of tetrazolium salts to their respective formazans is frequently used to determine the metabolic activity of cultured cells as an indicator of cell viability. For membrane-impermeable tetrazolium salts such as WST1 the application of a membrane-permeable electron cycler is usually required to mediate the transfer of intracellular electrons for extracellular WST1 reduction. Here we demonstrate that in addition to the commonly used electron cycler M-PMS, menadione can also serve as an efficient electron cycler for extracellular WST1 reduction in cultured neural cells. The increase in formazan absorbance in glial cell cultures for the WST1 reduction by menadione involves enzymatic menadione reduction and was twice that recorded for the cytosolic enzyme-independent WST1 reduction in the presence of M-PMS. The optimized WST1 reduction assay allowed within 30 min of incubation a highly reliable detection of compromised cell metabolism caused by 3-bromopyruvate and impaired membrane integrity caused by Triton X-100, with a sensitivity as good as that of spectrophotometric assays which determine cellular MTT reduction or lactate dehydrogenase release. The short incubation period of 30 min and the observed good sensitivity make this optimized menadione-mediated WST1 reduction assay a quick and reliable alternative to other viability and toxicity assays.
Asunto(s)
Astrocitos/química , Formazáns/química , Neuronas/química , Espectrofotometría , Vitamina K 3/química , Animales , Astrocitos/citología , Astrocitos/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Formazáns/análisis , Glioma/metabolismo , Glioma/patología , Humanos , Metosulfato de Metilfenazonio/análogos & derivados , Metosulfato de Metilfenazonio/química , Neuronas/citología , Neuronas/metabolismo , Octoxinol/química , Octoxinol/toxicidad , Oxidación-Reducción , Piruvatos/química , Piruvatos/toxicidad , Ratas , Ratas WistarRESUMEN
The pyruvate analogue 3-bromopyruvate (3-BP) is an electrophilic alkylator that is considered a promising anticancer drug because it has been shown to kill cancer cells efficiently while having little toxic effect on nontumor cells. To test for potential adverse effects of 3-BP on brain cells, we exposed cultured primary rat astrocytes to 3-BP and investigated the effects of this compound on cell viability, glucose metabolism, and glutathione (GSH) content. The presence of 3-BP severely compromised cell viability and slowed cellular glucose consumption and lactate production in a time- and concentration-dependent manner, with half-maximal effects observed at about 100 µM 3-BP after 4 hr of incubation. The cellular hexokinase activity was not affected in 3-BP-treated astrocytes, whereas within 30 min after application of 3-BP the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was inhibited, and cellular GSH content was depleted in a concentration-dependent manner, with half-maximal effects observed at about 30 µM 3-BP. The depletion of cellular GSH after exposure to 100 µM 3-BP was not prevented by the presence of 10 mM of the monocarboxylates lactate or pyruvate, suggesting that 3-BP is not taken up into astrocytes predominantly by monocarboxylate transporters. The data suggest that inhibition of glycolysis by inactivation of GAPDH and GSH depletion contributes to the toxicity that was observed for 3-BP-treated cultured astrocytes.